**6.2. CYP1A1 and CYP2B in different species after treatment with OC mixture**

56 New Approaches to the Study of Marine Mammals

**Figure 2.** (A-B): Basal levels of immunofluorescence (AUF/nucleus) of CYP1A1 (A) and CYP2B (B) in fibroblast cells of fin whale (B.p.), Bryde's whale (B.e.), sperm whale (P.m.), killer whale (O.o.), Risso's dolphin (G.g.), bottlenose dolphin (T.t.), striped dolphin (S.c.), long-beaked common dolphin (D.c.) and

common dolphin (D.d.).

Results of the mean levels of immunofluorescence of CYP1A1 (A) and CYP2B (B), revealed in cultured fibroblasts of different species treated with OC mixture and expressed as index numbers, are reported in Table 2A-B.


**Table 2.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with OC mixture. The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of these proteins. N.C. = no cells.

The results confirm the capability of this methodology to detect CYP1A1 (Table 2A) and CYP2B (Table 2B) induction with OC mixture in many species of this study; particularly we had induction of CYP1A1 and CYP2B, with respect to chemical blank (DMSO), at all doses in fin whale (RT2) (Figure 3A; D) and long-beaked common dolphin (MDC12); an induction of CYP1A1 was detected at all doses in Risso's dolphin (GGL1) and of CYP2B at all doses in sperm whale (PM6), killer whale (MOO12) and striped dolphin (RT1) (Figure 3F). No induction of CYP1A1 was detected in sperm whale (PM6) and bottlenose dolphin (TurNic), while CYP2B showed OC induction at least at one treatment dose in all species. Different induction responses were given by the different specimens: there was a dose/response induction for CYP1A1 only for long-beaked common dolphin (MDC12) and for CYP2B only for fin whale (RT2) (Figure 3D), while a bell-shaped response was present for CYP1A1 in Risso's dolphin (GGL1) and common dolphin (DDL1), and for CYP2B in sperm whale (PM6), killer whale (MOO12) (Figure 3E), striped dolphin (RT1) (Figure 3F) and common dolphin (DDL1). Discontinuous induction response was showed for CYP1A1 and CYP2B by the other specimens such as CYP1A1 in killer whale (MOO12) (Figure 3B) and striped dolphin (RT1) (Figure 3C). It is interesting to point out that all species, following treatment with the OC mixture, showed a greater response of CYP2B, compared to CYP1A1, confirming that these xenobiotics mostly with globular structure have a major ability to induce this cytochrome.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

*0.01 µg/ml 0.01 µg/ml 0.05 µg/ml 0.1 µg/ml* 

*0.01 µg/ml 0.01 µg/ml 0.05 µg/ml 0.1 µg/ml* 

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 59

**RT2 (fin whale)** 100 52.1 91.2 51.3 **MBE3 (Bryde's whale)** N.C. N.C. N.C. N.C. **PM6 (sperm whale)** 100 113.2 105.2 52.0 **MOO12 (killer whale)** 100 132.2 36.6 131.1 **GGL1 (Risso's dolphin)** 100 73.3 98.6 82.6 **TurNic (bottlenose dolphin)** 100 71.1 102.5 131.9 **RT1 (striped dolphin)** 100 128.4 232.6 273.1

**RT2 (fin whale***)* 100 60.2 68.4 54.1 **MBE3 (Bryde's whale)** N.C. N.C. N.C. N.C. **PM6 (sperm whale***)* 100 111.9 70.5 63.1 **MOO12 (killer whale)** 100 314.7 110.7 52.7 **GGL1 (Risso's dolphin)** 100 70.2 75.9 48.6 **TurNic (bottlenose dolphin)** 100 71.5 69.2 94.1 **RT1 (striped dolphin)** 100 177.4 109.9 128.3

**MDC12 (long-beaked common dolphin)** 100 205.2 127.6 189.4 **DDL1 (common dolphin)** 100 135.4 133.9 154.6

**MDC12 (long-beaked common dolphin)** 100 149.5 139.3 178.1 **DDL1 (common dolphin)** 100 118.4 85.8 324.5

**Table 3.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with flame retardants. The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of these

A dose dependent induction of CYP1A1 was detected in striped dolphin (RT1) and common dolphin (DDL1) and of CYP2B only in long-beaked common dolphin (MDC12). A bellshaped response was present for CYP1A1 and CYP2B in sperm whale (PM6), and for CYP2B

**6.3. CYP1A1 and CYP2B in different species after treatment with flame** 

*A CYP1A1 Nonane* 

*B CYP2B Nonane* 

Results of the mean levels of immunofluorescence of CYP1A1 (A) and CYP2B (B), revealed in cultured fibroblasts of different species treated with flame retardants and expressed as index numbers, are reported in Table 3A-B. Marked differences in CYP1A1 (Table 3A) and CYP2B (Table 3B) induction by flame retardants were detected in different species, with higher sensitivity of responses in striped dolphin (RT1) for CYP1A1 (Figure 4A) and killer whale (MOO12) for CYP2B. To be highlighted that we have an inductive response of both cytochromes in the same animals, precisely in sperm whale (PM6), killer whale (MOO12), striped dolphin (RT1) (Figure 4A; C), long-beaked common dolphin (MDC12) (Figure 4B; D) and common dolphin (DDL1). Bottlenose dolphin (TurNic) showed only the CYP1A1

**retardants** 

induction.

proteins. N.C. = no cells.

**Figure 3.** A-F: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-C) and CYP2B (D-F) in fibroblast cells of fin whale (RT2) (A, D), killer whale (MOO12) (B, E) and striped dolphin (RT1) (C, F) treated with OC mixture. DAPI and Alexa Fluor 594 (Intensity 200ms) images of DMSO and the five OC mixture treatments.
